MAJOR ARTICLE
Plasma Cytokine Levels and Risk of HIV Type 1 (HIV-1) Transmission and Acquisition: A Nested Case-Control Study Among HIV-1– Serodiscordant Couples Erin M. Kahle,1 Michael Bolton,6 James P. Hughes,2 Deborah Donnell,1,6 Connie Celum,1,3,4 Jairam R. Lingappa,3,4,5 Allan Ronald,8 Craig R. Cohen,7 Guy de Bruyn,9 Youyi Fong,6 Elly Katabira,10 M. Juliana McElrath,3,4,6 and Jared M. Baeten1,3,4; for the Partners in Prevention HSV/HIV Transmission Study Teama 1
Department of Epidemiology, 2Department of Biostatistics, 3Department of Global Health, 4Department of Medicine, 5Department of Pediatrics, University of Washington, and 6Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle; 7Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco; 8Department of Medicine, University of Manitoba, Winnepeg, Canada; 9Perinatal HIV Research Unit, University of Witwatersrand, Johannesburg, South Africa; and 10Infectious Disease Institute, Makerere University, Kampala, Uganda
Background. A heightened proinflammatory state has been hypothesized to enhance human immunodeficiency virus type 1 (HIV-1) transmission – both susceptibility of HIV-1-exposed persons and infectiousness of HIV-1infected persons. Methods. Using prospective data from heterosexual African couples with HIV-1 serodiscordance, we conducted a nested case-control analysis to assess the relationship between cytokine concentrations and the risk of HIV-1 acquisition. Case couples (n = 120) were initially serodiscordant couples in which HIV-1 was transmitted to the seronegative partner during the study; control couples (n = 321) were serodiscordant couples in which HIV-1 was not transmitted to the seronegative partner. Differences in a panel of 30 cytokines were measured using plasma specimens from both HIV-1–susceptible and HIV-1–infected partners. Plasma was collected before seroconversion for cases. Results. For both HIV-1–infected and HIV-1–susceptible partners, cases and controls had significantly different mean responses in cytokine panels (P < .001, by the Hotelling T2 test), suggesting a broadly different pattern of immune activation for couples in which HIV-1 was transmitted, compared with couples without transmission. Individually, log10 mean concentrations of interleukin 10 (IL-10) and CXCL10 were significantly higher for both HIV-1–susceptible and HIV-1– infected case partners, compared with HIV-1–susceptible and HIV-1–infected control partners (P < .01 for all comparisons). In multivariate analysis, HIV-1 transmission was significantly associated with elevated CXCL10 concentrations in HIV-1–susceptible partners (P = .001) and with elevated IL-10 concentrations in HIV-1–infected partners (P = .02). Conclusions. Immune activation, as measured by levels of cytokine markers, particularly elevated levels of IL-10 and CXCL1, are associated with increased HIV-1 susceptibility and infectiousness. Keywords.
HIV-1 acquisition; immune activation; Africa.
Immune activation, characterized by polyclonal B-cell activation, accelerated T-cell turnover, dendritic cell
Received 24 July 2014; accepted 31 October 2014; electronically published 10 November 2014. a Members of the study team are listed at the end of the text. Correspondence: Jared M. Baeten, MD, PhD, Department of Global Health University of Washington, 325 Ninth Ave, Box 359927, Seattle, WA 98104 (jbaeten@ uw.edu). The Journal of Infectious Diseases® 2015;211:1451–60 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected]. DOI: 10.1093/infdis/jiu621
depletion, and elevated proinflammatory cytokine levels, is a hallmark of human immunodeficiency virus type 1 (HIV-1) infection [1]. Immune stimulation by HIV-1 contributes to the depletion of CD4+ T cells, rather than to the purging of virus, and has been associated with accelerated progression of HIV-1 disease [2–5]. The dysregulation of cytokines, important markers of immune activation, has been shown to contribute to viral replication and disease progression [6, 7]. While the relationship between HIV-1 pathogenesis and persistent immune activation has been well described [1, 2], the role of systemic inflammatory response and Immune Activation and HIV-1 Transmission
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immune activation in HIV-1 transmission is less understood. Increased immune activation in persons infected with HIV-1 could result in increased viral replication that could facilitate HIV-1 infectiousness and onward transmission [8, 9], but no data have directly explored this hypothesis. Furthermore, for HIV-1–uninfected persons, immune activation and an elevated inflammatory response before infection has also been hypothesized to potentially heighten susceptibility and facilitate HIV-1 acquisition; however, few studies have directly compared systemic immune activation between those who acquire HIV-1 and those who are exposed but remain uninfected with HIV1. Naranbhai et al found significant association between markers of systemic immune activation, including cytokine concentrations, and HIV-1 acquisition among women [10], but few other data are available on the role of systemic immune function in HIV-1 acquisition or transmission. Insight into the roles of innate and adaptive immune functions and HIV-1 susceptibility and infectiousness is an important factor in the development of effective prophylactic and therapeutic HIV-1 vaccines, as well as strategies for HIV-1 prevention, such as preexposure prophylaxis and microbicides. Findings from the Step HIV-1 vaccine trial showed that, in a prime-boost HIV-1 vaccine strategy based on an adenovirus type 5 (Ad5) vector, there was a trend toward increased HIV-1 acquisition among Ad5-seropositive individuals. This finding indicated that the amnestic immune response elicited was harmful [11], suggesting a challenge for vaccine development and a critical need to better understand how immune activation affects HIV-1 transmission. In African populations, where both HIV-1 infection and other infectious diseases are frequently prevalent, chronic immune activation may be heightened by the presence of other infections, both systemic and mucosal, that could contribute to increased susceptibility and infectiousness [12]. The aim of the present study was to assess whether differences in markers of immune activation, as measured by a data from a panel of cytokines, were associated with an increased risk of HIV-1 transmission among heterosexual HIV-1–serodiscordant couples. Assessment of these factors in cases of virologically linked HIV-1 transmission in a prospective cohort of HIV-1– serodiscordant couples permitted simultaneous evaluation of the relationship between systemic immune activation and both the heightened susceptibility of HIV-1–uninfected partners and the infectiousness of HIV-1–infected partners. METHODS Study Population
We conducted a nested case-control study, using data from 2 prospective studies of African HIV-1–serodiscordant couples. Between November 2004 and April 2007, heterosexual HIV1–serodiscordant couples from 6 African countries (Botswana, Kenya, South Africa, Tanzania, Uganda, and Zambia) were 1452
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JID 2015:211 (1 May)
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Kahle et al
enrolled into the Partners in Prevention HSV/HIV Transmission Study, a randomized, double-blind, placebo-controlled clinical trial of herpes simplex virus type 2 (HSV-2) suppressive therapy to reduce HIV-1 transmission, as previously described [13]. HSV-2 suppressive therapy was found not to reduce HIV1 transmission within the partnerships [14]. In a parallel study at 2 sites (Kampala, Uganda, and Soweto, South Africa), HIV1–serodiscordant couples were enrolled into an observational study of immune correlates of HIV-1 protection (Couples Observational Study) [15]. In both studies, participants were ≥18 years of age and sexually active, and HIV-1–seropositive partners were not using antiretroviral therapy at the time of study entry. HIV-1–uninfected participants were followed up quarterly with HIV-1 serologic testing. Protection of Human Subjects
All participants received HIV-1 and risk-reduction counseling (both individually and as a couple), free condoms, and treatment for sexually transmitted infections (STIs), according to World Health Organization guidelines. Written informed consent was obtained from all participants. The study protocols were approved by the University of Washington Human Subjects Review Committee and ethical review committees at each of the study sites. Selection of Cases and Controls
Cases were couples in which HIV-1 was transmitted to the seronegative partner during the study, as confirmed by viral sequencing [16]. Cases consisted of 120 couples: 105 from the Partners in Prevention HSV/HIV Transmission Study and 15 from the Couples Observational Study. Controls were couples in which HIV-1 was not transmitted to the seronegative partner. Controls were selected randomly from each of the 2 contributing studies in proportion to research site and sex distribution to be representative of the entire cohort of nonseroconverting couples. In total, 321 controls were sampled (approximately 2.5:1 ratio of controls to cases). For both cases and controls, both the HIV-1–infected member and the partner who acquired HIV-1 infection (among cases) or the partner who remained uninfected (among controls) were included. Laboratory Testing
HIV-1 seroconversion of initially HIV-1–uninfected partners was confirmed by dual rapid HIV-1 antibody tests, enzyme immunoassay, Western blot, and plasma HIV-1 RNA detection. Plasma HIV-1 RNA levels for HIV-1–infected partners were quantified using the COBAS Ampliprep/COBAS TaqMan real-time HIV-1 RNA assay, version 1.0 (Roche Diagnostics, Indianapolis, Indiana). Serum was collected at quarterly study visits and archived at −80°C for subsequent laboratory testing. For the present analysis, archived blood plasma samples from cases were selected
from the visits immediately before the first evidence of HIV-1 infection (among cases, the initially HIV-1–uninfected partner had a negative result of a serological tests for HIV-1 and undetectable plasma HIV-1 RNA at this visit), to evaluate immune activation before HIV-1 transmission. Blood plasma specimens were collected for storage at quarterly visits, and the mean time between preseroconversion sample collection and HIV-1 infection among cases was three months. For controls, a single blood plasma sample per subject was selected, with the timing during follow-up selected so that the proportion of study months across controls was similar to visit months for the cases. Study visits from which control samples were selected occurred a mean of 6.5 months after enrollment. For both cases and controls, the mean time between sample collection and testing was 4 years. Samples were tested from both HIV-1–infected and uninfected partners. Immune activation was assessed from a standard panel of 30 cytokine analytes: T-helper type 1 cells (interferon γ [IFN-γ], interleukin 2 [IL-2], interleukin 7 [IL-7], interleukin 15 [IL-15], scd40l, Th2 cells (interleukin 4, interleukin 5, and interleukin 13), immunomodulatory cytokines (interleukin 1Rα, interleukin 10 [IL-10], interleukin 12p40 [IL-12p40], IL-12p70, and interleukin 17 [IL-17]), proinflammatory cytokines (IL-1α, IL-1β, interleukin 6, and CXCL8), chemokines (CCL11, CX3CL1, CXCL10, CCL3, CCL4, and tumor necrosis factor α [TNF-α]), and growth factors (epidermal growth factor [EGF], granulocyte colony-stimulating factor [G-CSF], granulocyte macrophage colony-stimulating factor, vascular endothelial growth factor, CCL2, and transforming growth factor α) [17]. Levels were measured using Luminex multiplex technology (Milliplex Human Cytokine/Chemokine panel, Millipore, Billerica, Massachusetts), and standard curves were analyzed using the nCal package (http://research.fhcrc.org/youyifong/en/resources/ncal. html) in the R statistical programming system [18]. The standard cytokine panel included potentially redundant and noninformative cytokines relative to our outcome of interest, but we included all cytokines from the panel for hypothesis-developing exploratory analysis of potential relationships between cytokines and HIV-1 risk. Plasma specimens were assayed in duplicate without dilution. Cytokine concentrations were measured in picograms per milliliter. Assays were performed blinded to case and control status. Statistical Analysis
Cytokine concentrations were log10 transformed, and results below the limit of quantification were assigned a value of half the limit of detection. The relationship between immune activation and HIV-1 susceptibility (using data from partners who were initially uninfected with HIV-1) and HIV-1 infectiousness (using data from HIV-1–infected partners) were initially analyzed separately. Hotelling T2 tests were performed to test for the global equality of mean cytokine concentrations between cases and controls. To assess differences in specific cytokine
concentrations, 2-sided Student t tests were used to compare mean concentrations of each of the 30 cytokine analytes between cases and controls. P values were adjusted for multivariate comparisons, using a permutation method, because standard methods for controlling for multiple comparisons are overly conservative when using highly correlated data (ie, interrelated cytokine concentrations) [19]. Cytokine analytes individually found to be significantly associated with seroconversion after controlling for multiple comparisons were then assessed as covariates in a multiple logistic regression (with case-control status as the outcome), with adjustment for important demographic, clinical, and behavioral predictors of HIV-1 risk in this population. Selected covariates were those determined to be associated with an increased risk of HIV-1 transmission and with differential cytokine concentrations. Confounders included in multivariate model were sex of the HIV-1 uninfected partner and, at the study visit selected for cytokine assessment, plasma HIV-1 RNA concentrations in the HIV-1–infected partner, report of unprotected sex in the partnership, and syndromic diagnosis of a STI, including urethritis, cervicitis, vaginitis, genital ulcer disease, pelvic inflammatory disease, genital herpes, or lymphogranuloma venereum, in either partner. Finally, we performed additional multivariate logistic regressions that included the cytokine concentrations for both partners, to assess the potential contribution of each cytokine to the risk of HIV-1 transmission within the partnership. To determine which, if any, cytokine concentrations were significantly associated with either HIV-1 transmission or acquisition risk, we used Spearman rank-order correlation and plotted the cytokine concentrations with a regression line. Analyses were performed using SAS v.9.3 (SAS Institute, Cary, North Carolina) and Prism graphing software (GraphPad Software, La Jolla, Carolina).
RESULTS Cytokine results were available for 441 HIV-1 serodiscordant couples (120 cases, and 321 controls). One HIV-1–uninfected control partner was excluded because their sample could not be analyzed. Most couples (93.2%) were married or living with their HIV-1–infected partner, and two-thirds were from eastern Africa (Table 1). A minority of individuals—33 HIV1–susceptible partners (10.3%) and 49 HIV-1–infected partners (15.3%)—had symptoms indicating an STI at the visit selected for cytokine testing. Compared with controls, cases were more likely to report unprotected sex (41.7% vs 19.0%; P < .001). For susceptible partners, those who acquired HIV-1 were more likely than those who remained HIV-1 uninfected to have a syndromic STI diagnosis at study visit (15.0% vs 4.7%; P = .002). Compared with HIV-1–infected control partners, HIV-1– infected case partners had higher median plasma HIV-1 RNA concentrations (4.9 vs 4.0 log10 copies/mL; P < .001).
Immune Activation and HIV-1 Transmission
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Table 1.
Characteristics of the Nested Case-Control Population, Overall and by Human Immunodeficiency Virus Type 1 (HIV-1) Status Couples With HIV-1 Acquisition, (n = 120)
Status, Characteristic
Couples Without HIV-1 Acquisition, (n = 321)
P Valuesa
Overall Female sex, HIV-1–infected partner
60 (50.0)
114 (35.5)
.006
East African (vs southern African)
79 (65.8)
219 (68.2)
.2
112 (93.3) 1 (0–2)
299 (93.2) 1 (0–2)
.5
Plasma Cytokine Levels and Risk of HIV Type 1 (HIV-1) Transmission and Acquisition: A Nested Case-Control Study Among HIV-1– Serodiscordant Couples Erin M. Kahle,1 Michael Bolton,6 James P. Hughes,2 Deborah Donnell,1,6 Connie Celum,1,3,4 Jairam R. Lingappa,3,4,5 Allan Ronald,8 Craig R. Cohen,7 Guy de Bruyn,9 Youyi Fong,6 Elly Katabira,10 M. Juliana McElrath,3,4,6 and Jared M. Baeten1,3,4; for the Partners in Prevention HSV/HIV Transmission Study Teama 1
Department of Epidemiology, 2Department of Biostatistics, 3Department of Global Health, 4Department of Medicine, 5Department of Pediatrics, University of Washington, and 6Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle; 7Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco; 8Department of Medicine, University of Manitoba, Winnepeg, Canada; 9Perinatal HIV Research Unit, University of Witwatersrand, Johannesburg, South Africa; and 10Infectious Disease Institute, Makerere University, Kampala, Uganda
Background. A heightened proinflammatory state has been hypothesized to enhance human immunodeficiency virus type 1 (HIV-1) transmission – both susceptibility of HIV-1-exposed persons and infectiousness of HIV-1infected persons. Methods. Using prospective data from heterosexual African couples with HIV-1 serodiscordance, we conducted a nested case-control analysis to assess the relationship between cytokine concentrations and the risk of HIV-1 acquisition. Case couples (n = 120) were initially serodiscordant couples in which HIV-1 was transmitted to the seronegative partner during the study; control couples (n = 321) were serodiscordant couples in which HIV-1 was not transmitted to the seronegative partner. Differences in a panel of 30 cytokines were measured using plasma specimens from both HIV-1–susceptible and HIV-1–infected partners. Plasma was collected before seroconversion for cases. Results. For both HIV-1–infected and HIV-1–susceptible partners, cases and controls had significantly different mean responses in cytokine panels (P < .001, by the Hotelling T2 test), suggesting a broadly different pattern of immune activation for couples in which HIV-1 was transmitted, compared with couples without transmission. Individually, log10 mean concentrations of interleukin 10 (IL-10) and CXCL10 were significantly higher for both HIV-1–susceptible and HIV-1– infected case partners, compared with HIV-1–susceptible and HIV-1–infected control partners (P < .01 for all comparisons). In multivariate analysis, HIV-1 transmission was significantly associated with elevated CXCL10 concentrations in HIV-1–susceptible partners (P = .001) and with elevated IL-10 concentrations in HIV-1–infected partners (P = .02). Conclusions. Immune activation, as measured by levels of cytokine markers, particularly elevated levels of IL-10 and CXCL1, are associated with increased HIV-1 susceptibility and infectiousness. Keywords.
HIV-1 acquisition; immune activation; Africa.
Immune activation, characterized by polyclonal B-cell activation, accelerated T-cell turnover, dendritic cell
Received 24 July 2014; accepted 31 October 2014; electronically published 10 November 2014. a Members of the study team are listed at the end of the text. Correspondence: Jared M. Baeten, MD, PhD, Department of Global Health University of Washington, 325 Ninth Ave, Box 359927, Seattle, WA 98104 (jbaeten@ uw.edu). The Journal of Infectious Diseases® 2015;211:1451–60 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected]. DOI: 10.1093/infdis/jiu621
depletion, and elevated proinflammatory cytokine levels, is a hallmark of human immunodeficiency virus type 1 (HIV-1) infection [1]. Immune stimulation by HIV-1 contributes to the depletion of CD4+ T cells, rather than to the purging of virus, and has been associated with accelerated progression of HIV-1 disease [2–5]. The dysregulation of cytokines, important markers of immune activation, has been shown to contribute to viral replication and disease progression [6, 7]. While the relationship between HIV-1 pathogenesis and persistent immune activation has been well described [1, 2], the role of systemic inflammatory response and Immune Activation and HIV-1 Transmission
•
JID 2015:211 (1 May)
•
1451
immune activation in HIV-1 transmission is less understood. Increased immune activation in persons infected with HIV-1 could result in increased viral replication that could facilitate HIV-1 infectiousness and onward transmission [8, 9], but no data have directly explored this hypothesis. Furthermore, for HIV-1–uninfected persons, immune activation and an elevated inflammatory response before infection has also been hypothesized to potentially heighten susceptibility and facilitate HIV-1 acquisition; however, few studies have directly compared systemic immune activation between those who acquire HIV-1 and those who are exposed but remain uninfected with HIV1. Naranbhai et al found significant association between markers of systemic immune activation, including cytokine concentrations, and HIV-1 acquisition among women [10], but few other data are available on the role of systemic immune function in HIV-1 acquisition or transmission. Insight into the roles of innate and adaptive immune functions and HIV-1 susceptibility and infectiousness is an important factor in the development of effective prophylactic and therapeutic HIV-1 vaccines, as well as strategies for HIV-1 prevention, such as preexposure prophylaxis and microbicides. Findings from the Step HIV-1 vaccine trial showed that, in a prime-boost HIV-1 vaccine strategy based on an adenovirus type 5 (Ad5) vector, there was a trend toward increased HIV-1 acquisition among Ad5-seropositive individuals. This finding indicated that the amnestic immune response elicited was harmful [11], suggesting a challenge for vaccine development and a critical need to better understand how immune activation affects HIV-1 transmission. In African populations, where both HIV-1 infection and other infectious diseases are frequently prevalent, chronic immune activation may be heightened by the presence of other infections, both systemic and mucosal, that could contribute to increased susceptibility and infectiousness [12]. The aim of the present study was to assess whether differences in markers of immune activation, as measured by a data from a panel of cytokines, were associated with an increased risk of HIV-1 transmission among heterosexual HIV-1–serodiscordant couples. Assessment of these factors in cases of virologically linked HIV-1 transmission in a prospective cohort of HIV-1– serodiscordant couples permitted simultaneous evaluation of the relationship between systemic immune activation and both the heightened susceptibility of HIV-1–uninfected partners and the infectiousness of HIV-1–infected partners. METHODS Study Population
We conducted a nested case-control study, using data from 2 prospective studies of African HIV-1–serodiscordant couples. Between November 2004 and April 2007, heterosexual HIV1–serodiscordant couples from 6 African countries (Botswana, Kenya, South Africa, Tanzania, Uganda, and Zambia) were 1452
•
JID 2015:211 (1 May)
•
Kahle et al
enrolled into the Partners in Prevention HSV/HIV Transmission Study, a randomized, double-blind, placebo-controlled clinical trial of herpes simplex virus type 2 (HSV-2) suppressive therapy to reduce HIV-1 transmission, as previously described [13]. HSV-2 suppressive therapy was found not to reduce HIV1 transmission within the partnerships [14]. In a parallel study at 2 sites (Kampala, Uganda, and Soweto, South Africa), HIV1–serodiscordant couples were enrolled into an observational study of immune correlates of HIV-1 protection (Couples Observational Study) [15]. In both studies, participants were ≥18 years of age and sexually active, and HIV-1–seropositive partners were not using antiretroviral therapy at the time of study entry. HIV-1–uninfected participants were followed up quarterly with HIV-1 serologic testing. Protection of Human Subjects
All participants received HIV-1 and risk-reduction counseling (both individually and as a couple), free condoms, and treatment for sexually transmitted infections (STIs), according to World Health Organization guidelines. Written informed consent was obtained from all participants. The study protocols were approved by the University of Washington Human Subjects Review Committee and ethical review committees at each of the study sites. Selection of Cases and Controls
Cases were couples in which HIV-1 was transmitted to the seronegative partner during the study, as confirmed by viral sequencing [16]. Cases consisted of 120 couples: 105 from the Partners in Prevention HSV/HIV Transmission Study and 15 from the Couples Observational Study. Controls were couples in which HIV-1 was not transmitted to the seronegative partner. Controls were selected randomly from each of the 2 contributing studies in proportion to research site and sex distribution to be representative of the entire cohort of nonseroconverting couples. In total, 321 controls were sampled (approximately 2.5:1 ratio of controls to cases). For both cases and controls, both the HIV-1–infected member and the partner who acquired HIV-1 infection (among cases) or the partner who remained uninfected (among controls) were included. Laboratory Testing
HIV-1 seroconversion of initially HIV-1–uninfected partners was confirmed by dual rapid HIV-1 antibody tests, enzyme immunoassay, Western blot, and plasma HIV-1 RNA detection. Plasma HIV-1 RNA levels for HIV-1–infected partners were quantified using the COBAS Ampliprep/COBAS TaqMan real-time HIV-1 RNA assay, version 1.0 (Roche Diagnostics, Indianapolis, Indiana). Serum was collected at quarterly study visits and archived at −80°C for subsequent laboratory testing. For the present analysis, archived blood plasma samples from cases were selected
from the visits immediately before the first evidence of HIV-1 infection (among cases, the initially HIV-1–uninfected partner had a negative result of a serological tests for HIV-1 and undetectable plasma HIV-1 RNA at this visit), to evaluate immune activation before HIV-1 transmission. Blood plasma specimens were collected for storage at quarterly visits, and the mean time between preseroconversion sample collection and HIV-1 infection among cases was three months. For controls, a single blood plasma sample per subject was selected, with the timing during follow-up selected so that the proportion of study months across controls was similar to visit months for the cases. Study visits from which control samples were selected occurred a mean of 6.5 months after enrollment. For both cases and controls, the mean time between sample collection and testing was 4 years. Samples were tested from both HIV-1–infected and uninfected partners. Immune activation was assessed from a standard panel of 30 cytokine analytes: T-helper type 1 cells (interferon γ [IFN-γ], interleukin 2 [IL-2], interleukin 7 [IL-7], interleukin 15 [IL-15], scd40l, Th2 cells (interleukin 4, interleukin 5, and interleukin 13), immunomodulatory cytokines (interleukin 1Rα, interleukin 10 [IL-10], interleukin 12p40 [IL-12p40], IL-12p70, and interleukin 17 [IL-17]), proinflammatory cytokines (IL-1α, IL-1β, interleukin 6, and CXCL8), chemokines (CCL11, CX3CL1, CXCL10, CCL3, CCL4, and tumor necrosis factor α [TNF-α]), and growth factors (epidermal growth factor [EGF], granulocyte colony-stimulating factor [G-CSF], granulocyte macrophage colony-stimulating factor, vascular endothelial growth factor, CCL2, and transforming growth factor α) [17]. Levels were measured using Luminex multiplex technology (Milliplex Human Cytokine/Chemokine panel, Millipore, Billerica, Massachusetts), and standard curves were analyzed using the nCal package (http://research.fhcrc.org/youyifong/en/resources/ncal. html) in the R statistical programming system [18]. The standard cytokine panel included potentially redundant and noninformative cytokines relative to our outcome of interest, but we included all cytokines from the panel for hypothesis-developing exploratory analysis of potential relationships between cytokines and HIV-1 risk. Plasma specimens were assayed in duplicate without dilution. Cytokine concentrations were measured in picograms per milliliter. Assays were performed blinded to case and control status. Statistical Analysis
Cytokine concentrations were log10 transformed, and results below the limit of quantification were assigned a value of half the limit of detection. The relationship between immune activation and HIV-1 susceptibility (using data from partners who were initially uninfected with HIV-1) and HIV-1 infectiousness (using data from HIV-1–infected partners) were initially analyzed separately. Hotelling T2 tests were performed to test for the global equality of mean cytokine concentrations between cases and controls. To assess differences in specific cytokine
concentrations, 2-sided Student t tests were used to compare mean concentrations of each of the 30 cytokine analytes between cases and controls. P values were adjusted for multivariate comparisons, using a permutation method, because standard methods for controlling for multiple comparisons are overly conservative when using highly correlated data (ie, interrelated cytokine concentrations) [19]. Cytokine analytes individually found to be significantly associated with seroconversion after controlling for multiple comparisons were then assessed as covariates in a multiple logistic regression (with case-control status as the outcome), with adjustment for important demographic, clinical, and behavioral predictors of HIV-1 risk in this population. Selected covariates were those determined to be associated with an increased risk of HIV-1 transmission and with differential cytokine concentrations. Confounders included in multivariate model were sex of the HIV-1 uninfected partner and, at the study visit selected for cytokine assessment, plasma HIV-1 RNA concentrations in the HIV-1–infected partner, report of unprotected sex in the partnership, and syndromic diagnosis of a STI, including urethritis, cervicitis, vaginitis, genital ulcer disease, pelvic inflammatory disease, genital herpes, or lymphogranuloma venereum, in either partner. Finally, we performed additional multivariate logistic regressions that included the cytokine concentrations for both partners, to assess the potential contribution of each cytokine to the risk of HIV-1 transmission within the partnership. To determine which, if any, cytokine concentrations were significantly associated with either HIV-1 transmission or acquisition risk, we used Spearman rank-order correlation and plotted the cytokine concentrations with a regression line. Analyses were performed using SAS v.9.3 (SAS Institute, Cary, North Carolina) and Prism graphing software (GraphPad Software, La Jolla, Carolina).
RESULTS Cytokine results were available for 441 HIV-1 serodiscordant couples (120 cases, and 321 controls). One HIV-1–uninfected control partner was excluded because their sample could not be analyzed. Most couples (93.2%) were married or living with their HIV-1–infected partner, and two-thirds were from eastern Africa (Table 1). A minority of individuals—33 HIV1–susceptible partners (10.3%) and 49 HIV-1–infected partners (15.3%)—had symptoms indicating an STI at the visit selected for cytokine testing. Compared with controls, cases were more likely to report unprotected sex (41.7% vs 19.0%; P < .001). For susceptible partners, those who acquired HIV-1 were more likely than those who remained HIV-1 uninfected to have a syndromic STI diagnosis at study visit (15.0% vs 4.7%; P = .002). Compared with HIV-1–infected control partners, HIV-1– infected case partners had higher median plasma HIV-1 RNA concentrations (4.9 vs 4.0 log10 copies/mL; P < .001).
Immune Activation and HIV-1 Transmission
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JID 2015:211 (1 May)
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1453
Table 1.
Characteristics of the Nested Case-Control Population, Overall and by Human Immunodeficiency Virus Type 1 (HIV-1) Status Couples With HIV-1 Acquisition, (n = 120)
Status, Characteristic
Couples Without HIV-1 Acquisition, (n = 321)
P Valuesa
Overall Female sex, HIV-1–infected partner
60 (50.0)
114 (35.5)
.006
East African (vs southern African)
79 (65.8)
219 (68.2)
.2
112 (93.3) 1 (0–2)
299 (93.2) 1 (0–2)
.5
